Building blocks of biofilms – an engaging and hands-on microbiology outreach activity for school children and the general public

Biofilms are naturally occurring communities of micro-organisms, attached to a surface and often embedded in a matrix of self-produced polymeric substances. Biofilms are widely implicated in human infections, particularly on prostheses and medical implants. Such biofilms are difficult to eradicate, often leading to replacement of the prosthesis and resulting in a significant burden to healthcare. Here we present a fun and engaging interactive activity targeted toward primary school/early secondary school children, introducing the concept of natural and healthcare-associated biofilms, using dental plaque as an archetypal example. Dental plaque forms as a result of poor oral/dental hygiene, and develops according to a typical series of defined stages: attachment and adherence to the surface, followed by colonization and maturation of the biofilm structure, and eventually, dispersal. This activity uses dental disclosing tablets to visualize real biofilms (plaque) on the participants teeth, and uses interlocking building-blocks to represent microorganisms, where children build three-dimensional ‘biofilms’ of varying shapes and structural integrities. Each of the stages of development are discussed in detail, and after building the biofilms, balls of different shapes, sizes and weights can be used as ‘antimicrobials’ to disrupt the biofilm structure. The outcomes of the activity are to enhance knowledge and general understanding of biofilms; their ubiquitous presence in the natural environment, development, implications in healthcare, and challenges of treatment. The various ‘antimicrobial’ balls also provide a basis to introduce and discuss drug selection for infections, and the importance of using the correct antimicrobial for different infections to avoid development of resistance.


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Biofilms are defined as aggregates of microorganisms (frequently as more than one type living together; 23 termed polymicrobial), often attached to a surface and embedded in a matrix of self-produced polymeric 24 substances. Their distribution in the natural environment is ubiquitous. They can exist on natural or 25 man-made abiotic (non-living) surfaces such as rocks, floors, walls etc., or on biotic (living) surfaces, 26 including wounds, teeth or skin/mucosal surfaces. Importantly, they can exist at the interface of both 27 abiotic and biotic surfaces, and in healthcare they are a significant burden both to the patient and the 28 health service resources when they colonise and infect a prosthesis such as an implant or replacement 29 joint.

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Approximately 60% of healthcare-associated infections are thought to have a biofilm origin, and they 32 can have some very substantial implications in our general health, particularly with oral hygiene. In 33 order to explain and discuss the concept of biofilms, we will be using the familiar example of dental 34 plaque as a familiar reference. After a period of restricted or poor oral hygiene, a plaque film begins to 35 develop on the tooth surface. This is the archetypal example of a biofilm -the structured community 36 of many different microbial species, attached to the tooth surface, and embedded in the self-produced 37 outer matrix, which follows the defined rules and sequence of development.

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Hundreds of different types of microorganisms exist in the mouth, and they can be found suspended in 42 their free-living state in saliva, known as the 'planktonic' state. Contrary to what was previously 43 thought, we now understand that the planktonic method of growth is not the preferred way for most 44 microorganisms to grow, but they tend to form biofilms. This is important for their survival, particularly 45 in the oral cavity where there is an ongoing risk of them washed away by the continual movement and 46 swallowing of saliva.

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Biotic (living) surfaces are rarely free of microbial matter. This is particularly true in the oral cavity,

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where the saliva provides a coating (pellicle) on the surfaces of the teeth and gums. This pellicle 50 contains a myriad of proteins and sugars, and makes it much easier for microorganisms to attach, as 51 there are far more receptors for them to bind to. Microorganisms have adapted over thousands (even 52 millions) of years to have the ability to attach to a range of surfaces and survive, and they do this through 53 very refined mechanisms, including forming biofilms which follow a distinct sequence of events for 54 formation detailed below. This pellicle not only supports a physical attachment, but the proteins and 55 sugars can also be used as a source of nutrients for the primary microbial cells that exist there.

Stages of biofilm development; attachment/adherence
58 Figure S1 details the stages of biofilm formation. Planktonic microbial cells are continually moving 59 around suspended in the saliva, and where they come into close proximity to the surface, weak attractive 60 forces come into play. Forces such as Van der Waals, electrostatic or ionic forces attract the microbial 61 cell toward the surface (Fig. S1a), where it can stick and eventually attach to specific receptors. Van 62 der Waals forces are a distance-dependent attraction/repulsion of two objects, based on interactions 63 between atoms or molecules. Similarly, electrostatic forces are non-contact forces but rely on 64 differences in charges between two objects to be attracted (to push away or pull towards each other).

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For example, a positively charged object is attracted to a negatively charged object, but will be 'pushed 66 away' or repelled from another positively charged object. Microbial cells have charges on their outer 67 surface, and this can influence whether they are attracted to or repelled from the surface. However, the 68 surface itself also has a charge, and depending on what is present on the surface can dictate the charge 69 this has, and therefore whether it attracts or repels other objects. These early attractive forces between 70 the microbe and the surface are relatively weak, so if there is, for example in the oral cavity, a strong 71 salivary flow, these microbial cells can become detached from the surface and remain in their planktonic 72 phase. However, shortly (approximately 60 minutes) after attachment, and in the absence of stronger 73 forces, the cells more strongly adhere to the surface using locking receptor interactions. This is the first 74 stage of biofilm formation.
The stronger binding mechanism of locking receptor interactions work similar to a hook and loop 76 concept. The microbial cell has proteins (ligands) on the outer surface known as adhesins, that stick out 77 looking for a suitable receptor to bind to. When it comes across something suitable, it will 'hook on' to 78 the receptor, making a much stronger binding/connection, which is deemed irreversible and not subject 79 to being washed away with the same forces as detailed above. Different microbes have different 80 adhesins that stick to different receptors, but an awareness of the hook and loop mechanism is sufficient 81 here.

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The first microbial cells to attach to the surface are often referred to as 'primary colonisers' -the first 85 to colonise the surface. These are typically those in highest abundance in the environment and those 86 that can exist in a range of environmental conditions. For example, Streptococcus bacteria are often 87 found during the early formation of dental plaque, and are known to be some of the first colonisers of 88 many oral surfaces. Having the primary colonisers attached to a surface now makes it much easier for 89 the eventual formation of a biofilm. Additional planktonic microbial cells not only need to attach to the 90 tooth surface with specific proteins to bind to, but have the opportunity to bind to other cells that have 91 already adhered to the surface (Fig. S1b). These cells are called 'secondary colonisers'. Many 92 microorganisms can interact with each other, either by producing chemical signals that they secrete into 93 the environment, or by direct contact. This direct contact interaction is similar to the primary coloniser 94 interacting with the surface and the bond is considered to be relatively strong, and is known as co-95 aggregation. Free-floating biofilms (floccules) also exist, and these are similar to surface-associated 96 biofilms, as they consist of co-aggregated cells and are often embedded in a matrix, but are not attached 97 to a specific surface. These have been found in environments such as streams and rivers, where the 98 water flow is too high for them to attach to the static surfaces, but they still maintain the ability to cluster 99 together in a biofilm form.

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The secondary colonisers may attach directly to the primary colonisers, resulting in a second layer of 102 cells still attached to the surface. This layering continues with more and more microbial cells attaching to the outermost layer, and as a result of cell multiplication from within the biofilm, until a multi-layered 104 structure is formed (Fig. S1c). This is the typical structure of a biofilm. A high-magnification 105 microscopy image of a polymicrobial biofilm grown under laboratory conditions illustrating this 106 structure is shown in Fig. S2.

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As the biofilm structure establishes and grows into this 3D structure, many of the cells produce 110 substances that contribute to building a protective matrix (Figs. S1c-d), such as secreted proteins, sugars 111 and DNA from the cells and environment. This provides a physical structure for the biofilm onto which 112 other cells can attach and establish themselves, further encouraging growth.

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The matrix has multiple purposes. It works as a physical scaffold for continued biofilm growth and 115 maturation into a 3D structure meaning other planktonic microorganisms can join the biofilm structure.

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It also provides physical protection from environmental fluctuations or extremes such as changes in pH, 117 temperature, nutrient availability, and acts as a physical barrier to treatments by antimicrobial 118 compounds. As biofilms are primarily made up of water, and rely on that for the survival of the 119 contained microorganisms, the matrix acts to stop desiccation in warmer and drier environments.

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The biofilm community works together for the greater good of survival, and there are various ways in 122 which the microorganisms can help each other for this to be achieved. Many microorganisms require 123 oxygen to survive (aerobic), but there are also many microorganisms that cannot survive in the presence 124 of oxygen (anaerobic). However, both of these are able to exist in the same biofilm in what seems to be 125 the same environmental conditions. This is possible through the formation of oxygen micro-gradients.

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Bacteria reliant upon oxygen consume the oxygen that is present, forming oxygen-limited environments 127 which then supports the life of those that require the absence or low levels of oxygen. This symbiotic 128 (mutually beneficial) relationship is one example of many similar relationships where some 129 microorganisms need certain nutrients or conditions that can only be produced by others in that community. In return, those that benefit from changes may produce factors that break down 131 antimicrobials or change different environmental conditions such as acidity.

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Within this protective biofilm environment, housed by the robust matrix, the biofilm cells continue to 135 develop into the 3D structure, whilst continuing to produce the matrix through to a mature biofilm state.

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The length of time this takes varies depending on the type of microorganisms that are growing. Dental 137 plaque can establish a relatively mature biofilm within a couple of days. However, environmental 138 biofilms may take weeks as they all require different nutrients and conditions to support their growth, 139 despite being highly adapted to their surroundings. Anaerobic microorganisms also tend to take a lot 140 longer to grow, as they are far slower in terms of growth than aerobic counterparts.

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Scientists understand that dispersal actually happens both actively (e.g. intentionally) as a result of

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During treatment and/or removal of biofilms from any surface, it is almost practically impossible to 154 remove every single microbial cell. This happens for a myriad of reasons, some of which we will discuss 155 here.

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Firstly, a biofilm is a complex community of microorganisms, many of which will have an inherent 158 ability to resist, withstand or tolerate antimicrobial treatment by chemicals, medication, or drugs. This 159 is due to several mechanisms in the cells, such as producing enzymes to break down the drugs, pumps 160 to actively pump the drug/chemicals outside of the microbial cell if they get in, a thick matrix 161 surrounding the biofilm itself to withstand penetration, differing pH within the biofilm structure, and 162 reduced oxygen concentration inside the matrix. All of these affect how the drug works and whether it 163 will be able to kill the cells or destroy/remove the biofilm structure.

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The microorganisms that are closest to the surface or in the middle of the 3D biofilm structure will also 166 undergo a process to reduce their metabolic activity, effectively going into a state of hibernation. Many 167 drugs work by the metabolism or growth of microorganisms, and so simply not growing or multiplying 168 is a great way to avoid being targeted by the drug. These are known as persister cells, and can stay in 169 this state of hibernation for a long time, beyond typical treatment periods. Then, when the conditions in 170 their local environment change to favour their growth, they will do so and have the ability, in the 171 absence of the drugs/chemicals, to grow and re-form a second biofilm, where the process continues.

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This activity aims to introduce the concept of antimicrobials as medications and treatments for 174 infections, through the use of soft balls. The balls are used by the participants to destroy the biofilm 175 structure, but those that stick to directly to the surface of the building block plate tend to do so very 176 strongly, and are not removed by these 'medicine balls'. These are the persister cells, which the 177 antimicrobial balls cannot remove (for reasons that can be discussed as detailed above, allowing the 178 participants to suggest or think about why that is). Additionally, introducing the concept of natural 179 acquisition of resistance can be done here. The generation time of microorganisms can be as short as 180 20 minutes, and with many new generations, come natural mutations in the DNA sequence of the 181 prodigy. Some of these mutations will be beneficial, and may infer a trait such as resistance to a drug.

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Then, when that particular drug is used to treat the biofilm, these inherently resistant microorganisms 183 will remain viable after the treatment, and then continue to grow and multiply, meaning the newly 184 formed structure will also be resistant to the previously used drug that wiped out the last community (after inheriting the same DNA as their 'parent cells'). This makes it much more difficult to treat 186 subsequent biofilms because there are fewer options of drugs that will be effective.

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It is important in this activity to complete the treatment steps in stages, which can be referred to as 198 'doses'. Each participant receives one 'antimicrobial ball', and they throw that at the structure. This is 199 the first dose. After this, the structural integrity is reviewed, and it will likely not have destroyed very 200 much. Then the participants complete a 'second dose', and then another review, and then a third dose 201 and so on. Each 'dose' will destroy more of the biofilm structure, which also relates to real-life 202 situations where several doses of a medication is required to have a sustained, cumulative effect.

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Not all bad news 205 Whilst we do tend to think of biofilms as being the bad guys of medicine, they deserve to be seen in a 206 negative light when they are healthcare-associated, but biofilms are also very useful in our daily lives.

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In fact, we have biofilms that live in our gut and help to break down foods; they are used in water and 208 wastewater treatment plants as a pre-treatment to feed on dead tissues or cells; and are involved in 209 bioremediation or waste breakdown. They are even used in the production of alcohol through 210 fermentation processes! So while there may be a lot of generalised negativity towards 'germs' and 211 biofilms, it is important to highlight the benefits of controlled biofilm use,.  -Insert disclosing tablet into mouth and chew whilst mixing around in saliva and washing around the mouth for approximately 30 seconds.
-Sip a small volume of water and rinse around mouth, then spit into waste container.
-Look at colouration of plaques on teeth and gum line.
-Pink colour indicates new plaque, blue/purple colour indicates older plaque.

Discussion points:
Dental plaque formation (e.g. biofilm), position of plaques on teeth, spread of plaques around front/back teeth and quantity. Brushing frequency, techniques and duration, and the need to brush with fluoride toothpaste (to reduce plaque bioburden and maintain enamel integrity).

Required materials:
-Large plastic interlocking building blocks (of varying shapes and sizes). A quantity of 50-100 per team is ideal.
-Building block base plates suitable for the blocks (2x) -Soft balls for destroying the biofilm structure (examples include plastic soft-play balls, soft cotton 'snow balls', table tennis balls etc. Dense but soft balls such as the snow balls are preferred and have been evaluated for suitability.

Method:
-Begin by marking out and explaining the area in which the participants will be building their biofilms. Approximately 10 x 10 pegs is normally suitable for this activity.
-Explain briefly the typical process of biofilm formation to the participants, using blocks to act as different microbes (and the Methods Poster (Appendix 4) as a reference): o Start with freely floating primary colonisers that stick to the surface. Attach a number of these to the pegs.
o Secondary colonisers then bind to the already attached primary colonisers. It is important to emphasise that the microbes never typically bind covering the whole surface (in this case avoiding complete coverage of the blocks), but they attach to the edges/outermost block surfaces. For example, on a 2x2 block, another block would cover 1x2 pegs, leaving the others free. The next block would then either attach to the remaining 1x2 pegs, or similarly, 1x2 on the second layer block. See image below.
o It is also necessary to emphasise the importance of channels within the biofilms. These channels are essential for nutrient and gaseous transfer into and within the biofilm structure, and therefore needs to be represented in this biofilm structure. The participants are not to build a tower-like structure, with impenetrable outer 'walls', but to allow channels for these nutrients to pass in and around, to support the life and growth of microbes deep within the biofilm depths and closest to the surface.
-Split the participants into two teams, each with a board and a set quantity of building blocks. If there are many participants, consider sub-splitting these into different groups for the following approach.
-Allow the participants to build the biofilms in a given time period (e.g. 1-2 minutes, but to be guided by the speed in which they are building successfully). If teams are too large, and need to be split into smaller groups within the teams, assign these a number or letter (e.g. Team 1 group A, Team 1 Group B etc.). Allow 30-60 seconds for each group to build the biofilm structure, after which the groups switch places, and the next group continues to build on the previous structure. This is continued until each group has contributed to the structure, and/or the overall time allowance has elapsed. Any unused blocks are counted but removed from the area.
-The biofilm structure is quantified by number of blocks used (or by subtraction of those not used), and then the educator can discuss the structure of the biofilms. Highlight the typical characteristics of the biofilm such as shape, height and presence of channels.
-The teams switch to the other team's biofilm, and are given the antimicrobial balls in preparation for destroying the structure. This is to be gauged on number of participants, but ideally 1-3 attempts to destroy per participant is normally sufficient.
-Allow the participants to throw the balls against the biofilm structure, and destroy as much as they can before switching to the other team and other biofilm structure. Count the number of blocks (microbes) that remain attached to the surface, and calculate percentage removal of biofilm mass. The team that removes the highest percentage of biofilm structure is considered the winner.
-This can be repeated with alternative 'antimicrobial balls', or if discussed prior to the activity, the participants can choose their favoured 'antimicrobial ball' from an available range for the type of infection the educator wants to portray.